Fig 2: TPD54 affects cellular sensitivity to metformin treatment accompanied by altered ROS production in MCF7 cells and breast cancer patient-derived xenografts. a TPD54 protein level was detected by western blot in control and TPD54 transiently knocked down cells treated with or without 8-mM metformin for 2 days. C: 1× PBS, M: metformin b TPD54 protein level was detected by western blot in control and TPD54 stably knocked down MCF-7 cells treated with or without 8-mM metformin for 2 days. C: 1× PBS, M: metformin. c Percentage of dead cells was plotted in cells transfected with control and TPD54 siRNAs and treated with metformin at indicated concentrations for 2 days. d Percentage of dead cells was plotted in control and TPD54 stably knocked down cells treated with metformin at the indicated concentrations for 2 days. e Fold changes of Mitosox mean fluorescence intensity representing mitochondrial ROS production was quantified in cells described in c. f The same assays and quantifications were performed in cells described in d. g Western blot analysis was performed using lysates from different breast cancer xenospheroids to detect TPD54. 3D cell viability was measured for xenospheroids treated with metformin at the following concentrations for 5 days: 8, 16, and 32 mM metformin. h TPD54 was overexpressed in BJ43 and treated with metformin as indicated for 3 days. 3D viability was performed. All data presented are representative experimental data that have been replicated by independent experiments and are reported as the mean ± SD from triplicate data of one experiment. Values are presented as mean ± SD, student t test was used to calculate p value for two-group comparisons with ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05)

Fig 3: TPD54 knockdown leads to increased PDH E1α phosphorylation at serine 232 by PDK1 followed by increased ubiquitination and degradation. a PDH E1α protein levels were detected by western blot in control and TPD54 stably knocked down cells with either DMSO or MG132 (10 μM) treatment for 1 day. b control and TPD54 stably knocked down cells transfected with control siRNA or siPDK1 were treated with metformin for 2 days. Western blot assays were performed to detect indicated proteins. c The ratio of pS232 PDH E1α to total PDH E1α in Fig. 5b was quantified and plotted as a bar graph. d Immunoprecipitation (IP) of PDH E1α with lysate from control cell and TPD54 stably knocked down cells was performed followed by immunoblotting of indicated proteins. Input represents whole cell lysate. e immunoprecipitation (IP) of PDH E1α with lysate of TPD54 stably knocked down cells with or without transient PDK1 knockdown was performed followed by immunoblotting of indicated proteins. Total proteins were determined in the input. f Control and TPD54 stably knocked down cells were transiently transfected with control siRNA or SiPKD1, followed by metformin treatment for 2 days. Western blot was then performed to detect indicated proteins. g Cell survival was determined in cells transfected with control, siPDK1, shTPD54, or double knockdowns. Values are presented as mean ± SD, student t test was used to calculate p value for two-group comparisons at different concentrations of metformin treatment (shCon siCon vs. shCon siPDK1 or shTPD54 siCon vs. shTPD54 siPDK1 with ***p < 0.001, **p < 0.01, *p < 0.05). h Western blot was performed to detect transient transfected WT PDH E1α and PDH E1α S232A mutant using SNAP-tag antibody and its ubiquitination modification after immunoprecipitation PDH E1α in control cells or TPD54 stably knocked down cells. Ubiquitination status and PDH E1α in whole cell lysate was detected by western blot as demonstrated in Input. i Western blots detecting protein changes among wild-type PDH E1α and its mutants S232A, S293G, and S300A were performed using SNAP-tag antibody in MCF-7 cells with or without transient TPD54 knockdown. All data presented were representative experimental data that have been replicated by independent experiments

Fig 4: TPD54 interacts with pyruvate dehydrogenase and plays an important role in breast cancer cells. a Images and the number of colony formation in control and stable TPD54 knockdown MCF-7 cells are presented. b Images and the number of colony formation of vector control and TPD54 overexpressed MCF7 cells are shown. Data were reported as the mean ± SD from triplicate data of one experiment. Values are presented as mean ± SD, student t test was used to calculate p value for two-group comparisons with ***p < 0.001 and *p < 0.05).c Endogenous TPD54 protein was detected using immunofluorescence. Mitochondria were labeled by DsRed Mito through transient transfection. The nucleus was labeled by DAPI staining. Images were taken at 200×. d Fractionations of cytoplasm (Cyto) and mitochondria (Mito) were performed in MCF-7 cells. Western blots were performed using cytoplasmic and mitochondrial fractions to identify TPD54 subcellular localization. Vinculin and NDUFB8 were used as cytoplasmic and mitochondrial markers and loading controls. e Endogenous TPD54 immunoprecipitation was performed in MCF-7 cells. Western blot detected TPD54 pulldown and its associated proteins in PDH complex. WL represents whole cell lysate as input. f PDH E1α and TPD54 were detected in MCF7 and MDA-MB-231 cells using immunofluorescence. The nucleus was labeled by DAPI staining. Images were taken at 100×. All data presented were representative experimental data that have been replicated by independent experiments

Fig 5: Proposed model of the mechanisms of TPD54 in regulation of PDH E1α in breast cancer cells and its effect on metformin response. TPD54 interacts with pyruvate dehydrogenase complex and affects PDH protein and enzyme activity. Loss of TPD54 causes increased phosphorylation of PDH E1α at S232 by PDK1, resulting in further ubiquitination and protein degradation of PDH E1α, and this phenotype can be restored by PDK1 knockdown (b, c). Loss of TPD54 leads to reduced oxidative phosphorylation and ROS production, which contribute to reduced metformin cytotoxicity (a, b). This can be reversed by activating pyruvate dehydrogenase by knocking down upstream inhibitory kinases PDK2 (b, c)